BACKGROUND Currently, nanobodies are undergoing intensive studies due to their characteristics of nanoscale size, high affinity and specificity, and deep tissue penetration. The development of novel and simplified methods to purify nanobodies is required because high purity and high recovery are the limiting factors for the downstream preparation of nanobodies. RESULTS In this study, ammonium sulfate precipitation (ASP) and electropositive multimodal chromatography (Capto MMC) were integrated for tag-free nanobody (2D5) purification. ASP was our first choice for protein purification because the process is simple, easy to operate, and has low cost. ASP achieved a 36% reduction in Escherichia coli host cell proteins (HCPs), a 99.1% reduction in E. coli host cell DNA, and an 85.1% reduction in endotoxins with a recovery of 81.7% and a purity enhancement from 6.2% to 73.9%. Capto MMC was seamlessly integrated for 2D5 purification. The recovery of this step was 83.0%. The entire two-step purification platform yielded 2D5 with 397 ppm of HCPs, 198 ppb of DNA, 204 EU/mL of endotoxins, and total protein recovery of 67.8%. The purity of 2D5 was 97.5%. The purification method was also used to purify a nanobody against PD-L1 (KPU) with 97.7% purity, > 95.7% recovery, 223 ppm of HCPs, 634 ppb of DNA and 154 EU/mL of endotoxins. CONCLUSION The established method had no effect on the affinity of 2D5. A simple and efficient purification platform that integrates ASP and Capto MMC was established, providing a more promising alternative to the conventional affinity chromatography-based nanobody purification strategy. (c) 2019 Society of Chemical Industry